Fungal cellulases

ABSTRACT

The present invention relates to peptides with multiple enzymatic activitives: carboxymethyl cellusase(CMCase), endoglucanase and β-glucanase, corresponding nucleotides, vectors and transformed hosts, methods of preparation and use. The peptide is used in the food industry for the liquefaction of plant cell material, e.g., in vegetable or fruit juice manufacture, or as processing aids to reduce the fouling of membranes.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to novel enzymes. More specifically, theinvention relates to novel fungal cellulases and methods of obtainingthe DNA encoding them.

BACKGROUND

There is an increasing interest in glucanases in the food and feedindustries. These enzymes find application for instance in the fruitjuice industry for liquefaction of plant cell wall material (pendingapplication EP 94202442.3). They may also serve as processing aids toreduce fouling of membranes. In the feed industry the role ofβ-glucanases in reducing the viscosity of various sorts of grains iswell established. Many of the enzyme preparations used in the food andfeed area are derived from Aspergillus species, usually Aspergillusniger. This is a safe host which produces a large variety of enzymessuch as pectinases and hemicellulases with characteristics which makethem suitable for applications at moderate temperatures and at neutralto acidic pH. In contrast to pectinases and hemicellulases, cellulasesare usually derived from Trichoderma. Trichoderma species such asreesei, viride or longibrachiatum are good producers of cellulolyticenzymes. However, Trichoderma enzymes cannot be used everywhere due toregulatory constraints. Thus, it would be of considerable economic valueto have a good source of Aspergillus enzymes. However, up till now, ithas not been possible to clone the genes encoding glucanases from A.niger using the traditional method involving enzyme purification,partial amino acid sequencing and isolation of the gene or cDNA for theenzyme of interest by the derived DNA sequence.

SUMMARY

The present invention provides polypeptides with cellulase activity,corresponding DNA sequences, vectors, transformed hosts and a method forthe production of these polypeptides which are obtainable fromAspergillus niger var. niger or Aspergillus niger var. tubigensis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows viscosity reduction due to CMCase activity of Aspergillusniger cellulases.

FIG. 2 shows viscosity reduction due to β-glucanase activity ofAspergillus niger cellulases.

DETAILED DESCRIPTION

The present invention relates to novel fungal cellulases and to methodsof identifying the DNA which encodes these fungal cellulases.

In the method according to the invention, fungal cellulases are clonedby expression cloning using a cDNA library in the form of prokaryotichost cells which have been transformed with DNA which is obtainable fromfungi. Screening of the host cells is performed after the cDNA has beeninserted into a plasmid. The prokaryotic host cells are preferablybacterial cells, more preferably E. coli.

In the present context, the term ‘cellulase’ refers to an enzyme whichdegrades carboxymethyl cellulose (CMC) and/or β-1,3-glucan and/or β-1,4glycan and/or xyloglucan.

In contrast to existing methods, the method according to the inventionis quick, straightforward and efficient. Surprisingly high numbers ofcellulase-positive clones are found by screening 10²-10⁴ clones, ratherthan 10⁵-10⁶ which is the case for existing methods (Dalbøge &Heldt-Hansen (1994) Mol. Gen. Genet. 243: 253-260).

Another surprising advantage of the method is that in contrast to mostexisting colony screening methods, there is no need to subsequently lysebacterial colonies to release their contents, including possible geneproducts of interest.

Expression cloning according to the invention may be carried out inprokaryotic cells, there is no need to transfer genetic material into aneukaryotic organism anywhere during the cloning procedure.

The cDNA library is prepared from the mRNA of a fungus of interest.Examples of fungi of particular interest are those of the genusAspergillus, more specifically Aspergillus niger var. niger or A. nigervar. tubigensis.

The mRNA which is used to prepare cDNA may be constitutively present orits expression may be induced. The method according to the invention isso effective that a large percentage of colonies will produce thecellulase of interest.

The library is constructed in a vector. This vector may be any vehiclesuitable for the transfer and/or expression of genetic material,including plasmid and phage vectors.

In a preferred embodiment, the library is constructed in a phage vector,preferably a ZAP™ vector or a derivative thereof, more preferably λUni-ZAP™ XR. The primary library which is obtained in this way isamplified using a suitable host cell, preferably E. coli, morepreferably E. coli XL1 Blue MRF′.

The phages are then converted into phagemids, preferably bysuperinfection with a filamentous helper phage and an E. coli hoststrain, such as E. coli SOLR. In this way a double stranded phagemid iscreated. Herein, the term ‘phagemid’ refers to a phage genome which hasbeen converted into a plasmid. Since all plaques are converted intophagemids prior to screening, clones showing cellulase activity with lowmRNA level are not mistakenly rejected as negative for activity. Hence,it will be clear to the skilled addressee that screening in the plaquestage is superfluous.

In the identification method according to the invention, DNA encodingcellulases is cloned by screening for expression of the cellulase, i.e.cellulase activity, rather than by going through the tedious tasks ofpurifying the protein, amino acid sequencing and molecular cloning ofthe gene. One technique of expression cloning is a plate assay, whereina cDNA library is plated on to a medium of a composition which enablesscreening for cellulase-positive colonies. Screening for cellulases doesnot require pure protein, only the availability of a suitable assay forthe detection of cellulase activity. This may be a standard assay; forexample, cellulase activity may be detected using an overlay containingcarboxymethyl celullose, followed by visualisation by staining withCongo Red. However, any other kind of assay which allows theidentification of DNA by expression of the protein it encodes may beused in the method according to the invention.

Alternatively, a purpose-made assay may be developed for the detectionof protein activity, for example where a suitable standard assay is notavailable.

In addition to cellulases, other enzymes may be detected, such asamylases, arabinoxylan degrading enzymes, catalases, galactanases,lipases, oxidases, pectinases, phosphatases, proteases and xylanases.Activity of these enzymes may be detected using methods known in theart.

The present invention also provides isolated nucleic acid fragmentscomprising a sequence encoding a polypeptide of the invention, i.e., acellulase obtainable from Aspergillus niger var. niger or A. niger var.tubigensis.

The nucleic acid fragments of the invention may comprise DNA or RNA,preferably DNA. Two preferred DNA fragments of the invention are thosewhose nucleic acid sequences are given in SEQ ID. No. 1 and 3. Thenucleic acid fragments of the invention are not, however, limited tothese preferred fragments. Rather, the invention also encompassesvariants of SEQ ID No. 1 and 3 obtainable from Aspergillus niger var.niger or Aspergillus niger var. tubigensis encoding polypeptides havingcellulase activity. For example, therefore, the invention providesdegenerate variants of SEQ ID. No. 1 or 3 that encode the polypeptidesof SEQ ID No. 2 or 4. Similarly, such variant nucleic acid fragments maycode for variants of the polypeptides of SEQ ID. No. 2 or 4, whichvariant polypeptides differ from SEQ ID NO. 2 or 4 by the deletion,insertion or substitution of one or more amino acids, as long as thedeletion, insertion or substitution does not abolish the cellulaseactivity of the polypeptide. Thus, the variant polypeptides of theinvention retain some or all of the activity, typically substantiallythe activity, of the polypeptide of SEQ ID NO. 2 or 4.

Also, the invention provides variant isolated nucleic acid, preferablyDNA, fragments having a high degree of sequence identity with thenucleic acid sequences of SEQ ID. No. 1 or 3. Thus they are typicallysubstantially homologous to SEQ ID. NO. 1 or 3. Typically, such variantfragments have at least 90% sequence identity with SEQ ID No. 1 or 3.Similarly, such variant fragments may differ from SEQ ID No. 1 or 3 bythe deletion, substitution or insertion of one or more amino acids, aslong as the deletion, substitution or insertion does not abolish thecellulase activity of the encoded polypeptide. Thus, the encodedpolypeptide retains some or all of the cellulase activity of thepolypeptide of SEQ ID No. 2 or 4.

Variant nucleic acid fragments of the invention may be obtained from anyorganism, although they are preferably obtained from fungi or yeasts,more preferably fungi of the genus Aspergillus, most preferably A. nigeror A. tubigensis.

The invention also provides recombinant nucleic acids, preferably DNA,comprising the nucleic acid fragments of the invention. Typically, theseare in the form of recombinant nucleic acid vectors.

A vector of the invention may be of any type known in the art, and maycomprise DNA or RNA, as appropriate. For example, the construct may bein linear or circular form. Plasmids are one preferred type of vector.Vectors of the invention may be cloning vectors; these are useful inmultiplying the nucleic acid fragments of the invention, and in theisolation and identification of nucleic acids of the invention. Vectorsof the invention may also be expression vectors; these are useful foridentifying nucleic acids of the invention by expression cloning, andfor producing polypeptides of the invention. The same vector can act,when appropriate, as a cloning vector and an expression vector forexpression cloning, as described herein for λ phage vectors.

Those of skill in the art will be able to prepare suitable vectors ofthe invention starting with widely available vectors which will bemodified by genetic engineering techniques known in the art, such asthose described by Sambrook et al (Molecular cloning: a LaboratoryManual; 1989).

A vector according to the invention typically comprises one or moreorigins of replication so that it can be replicated in a host cell, suchas a bacterial, yeast or fungal cell (this enables constructs to bereplicated and manipulated, for example in E. coli, by standardtechniques of molecular biology). A vector, especially an expressionvector, also typically comprises at least the following elements,usually in a 5′ to 3′ arrangement: a promoter for directing expressionof the nucleic acid sequence and optionally a regulator of the promoter,a transcription start site, a translational start codon, and a nucleicacid sequence of the invention.

The vector may also contain one or more selectable marker genes, forexample one or more antibiotic resistance genes. Such marker genes allowidentification of transformants. Optionally, the construct may alsocomprise an enhancer for the promoter. The vector may also comprise apolyadenylation signal, typically 3′ to the nucleic acid encoding thefunctional polypeptide. The vector may also comprise a transcriptionalterminator 3′ to the sequence encoding the polypeptide of interest.

The vector may also comprise one or more introns or other non-codingsequences, for example 3′ to the sequence encoding the polypeptide ofthe invention.

In a typical vector, the nucleic acid sequence of the invention isoperably linked to a promoter capable of expressing the sequence.“Operably linked” refers to a juxtaposition wherein the promoter and thenucleic acid sequence encoding the polypeptide or protein are in arelationship permitting the coding sequence to be expressed under thecontrol of the promoter. Thus, there may be elements such as 5′non-coding sequence between the promoter and coding sequence. Suchsequences can be included in the vector if they enhance or do not impairthe correct control of the coding sequence by the promoter.

Any suitable promoter that is capable of directing expression of thenucleic acid encoding the polypeptide of the invention may be includedin the vector. For example, the promoter may be a bacterial, eukaryoticor viral promoter. The promoter may be constitutive or inducible.

The invention also provides recombinant host cells harbouringrecombinant nucleic acids of the invention, either integrated into thehost cell genome or free in the cell. The host cell may be of anysuitable type. For example the host cell may be a bacterial (e.g. E.coli), yeast (e.g. K. lactis), fungal (e.g. Aspergillus), animal (e.g.insect or mammalian) or plant cell. Bacterial host cells are, forexample, useful in the expression cloning of DNA fragments of theinvention. Bacterial and other cell types may be useful in producingpolypeptides of the invention.

The recombinant nucleic acid of the invention may be introduced into thehost cell by any means known in the art. For example, the host cell maybe transformed or transfected by any suitable method, such as themethods disclosed by Sambrook et al (Molecular cloning: A LaboratoryManual; 1989). For example, recombinant nucleic acids comprising nucleicacid sequences according to the invention may be packaged intoinfectious viral particles, such as retroviral particles. Therecombinant nucleic acids may also be introduced, for example, byelectroporation, calcium phosphate precipitation, lipofection, biolisticmethods or by contacting naked recombinant nucleic acids with the hostcell.

The invention also provides polypeptides encoded by the nucleic acids ofthe invention. These polypeptides have cellulase activity, as definedherein. Two preferred polypeptides of the invention are those whosesequences are given in SEQ ID NO. 2 and 4. However, the invention is notlimited to these sequences; rather, it encompasses all polypeptidesencoded by the nucleic acid fragments of the invention, that havecellulase activity, as defined herein.

In particular, the invention provides variant polypeptides havingsequences related to those of SEQ ID NO. 2 and 4. Typically, suchvariants have a high degree of sequence identity with SEQ ID NO. 2 or 4,for example at least 70% sequence identity, thus, they are typicallysubstantially homologous to the polypeptides of SEQ ID NO. 2 or 4.Similarly, variant polypeptides of the invention may differ from SEQ IDNO. 2 or 4 by the deletion, insertion or substitution of one or moreamino acids, as long as the deletion, insertion or substitution does notabolish the cellulase activity of the polypeptide. Thus, the variantpolypeptides of the invention retain some or all of the activity,typically substantially the activity, of the polypeptide of SEQ ID NO. 2or 4.

Polypeptides of the invention may be in isolated form. For example theymay be substantially or completely isolated.

The invention also provides methods of producing polypeptides of theinvention. These comprise culturing host cells of the invention underconditions that permit the expression of polypeptides of the inventionfrom the recombinant nucleic acids of the invention; and, optionally,recovering the polypeptide thus produced.

The polypeptide may be recovered by any suitable means known in the art.

Utility

Cellulases according to the invention may be used, alone or incombination with other enzymes, in the food industry, e.g. for theliquefaction of plant cell material, e.g. in vegetable or fruit juicemanufacturing, or as processing aids to reduce fouling of membranes.

The cellulases may also be used in the feed industry. Examples ofapplications are their use for the improvement of feed utilization bybreaking down cell walls and or reducing the viscosity of various kindof grains.

Another application is in the textile industry for the treatment of bothwoven and knitted fabric, e.g. to achieve quality improvement or specialeffects (worn look).

Yet another application is in the detergent industry, e.g. in adetergent composition. The detergent composition may be formulated inany convenient form, such as powder, liquid, etc. The detergentcomposition may contain one or more other enzymes and other ingredientsknown in the art, such as builders, bleaching agents, perfumes etc.

The cellulases according to the invention may also be used in the pulpand paper industry for biopulping and biobleaching.

Experimental

Standard recombinant DNA technology such as bacterial growth, DNAisolation, hybridisation, restriction enzyme digestion and DNAsequencing are according to Sambrook et al. (1989): Molecular cloning, alaboratory manual, Cold Spring Harbor Laboratory Press, New York.).

EXAMPLES Example I Construction of Aspergillus niger cDNA ExpressionLibrary in E. coli

Example I.1

Induction and Isolation of mRNA

A. niger N400 cultures were grown for 69 and 81 h respectively, asdescribed in EP-A-0 463 706 without yeast extract and with 2% crudewheat arabinoxylan fraction instead of oat spelt xylan, after which themycelium was harvested by filtration and then washed with sterilesaline. The mycelium was subsequently frozen in liquid nitrogen afterwhich it was powdered using a Microdismembrator (Braun). Total RNA wasisolated from mycelial powder in accordance with the guanidiumthiocyanate/CsCl protocol described in Sambrook et al. (1989), exceptthat the RNA was centrifuged twice using a C₅Cl gradient. Poly A⁺ mRNAwas isolated from 5 mg of total RNA by oligo (dT)-cellulosechromatography (Aviv and Leder, 1972, Sambrook et al., 1989) with thefollowing modifications: SDS is omitted from all solutions and theloading buffer was supplemented with 9% (v/v) dimethylsulfoxide.

Example I.2

Construction of the cDNA Library

cDNA was synthesized from 7 μg poly A⁺ mRNA and ligated intobacteriophage lambda λ Uni-ZAP XR using the ZAP™-cDNA synthesis kit(Stratagene) according to the manufacturer's instructions. Afterligation of the cDNA into Uni-ZAP XR vector-arms, the phage DNA waspackaged using Packagene™ extracts (Promega) according to themanufacturers instructions. Ligation of 120 ng cDNA in 1.2 μg vectorarms and subsequent packaging of the reaction mixture resulted in aprimary library consisting of 3.5×10⁴ recombinant phages. This primarylibrary was amplified using E.coli XL1-Blue MRF′, titrated and stored at4° C.

Example I.3

Conversion of Phages Into Phagemids

Phages were propagated by plating them in NZYCM topagarose containing0.7% agarose on 85 mm diameter NZYCM (1.5% agar) plates as described byManiatis et al. (Maniatis et al. (1982): Molecular cloning, a laboratorymanual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. pp64)using E. coli BB4 as plating bacteria. After overnight incubation at 37°C. confluent plates were obtained from which the phages were eluted byadding 5 ml SM buffer and storing the plate for 2 hrs at 4° C. withintermittent shaking. After collection of the supernatant, the bacteriawere removed from the solution by centrifugation at 4.000×g at 4° C. for10 min. To the supernatant, 0.3% chloroform was added and the number ofplaque forming units (pfu) was determined. The phage stock containedapproximately 10¹⁰ pfu/ml.

The recombinant Uni-ZAP XR clones containing A. niger cDNA wereconverted to Bluescript phagemids using superinfection with thefilamentous helper phage EXASSIST™ and E. coli SOLR strain which areincluded in the cDNA synthesis kit from Stratagene, according to themanufacturer's instructions. For long term storage a glycerol stockcontaining about 100 colonies per μl of suspension was stored at −80° C.

Example II Screening of a Plasmid cDNA Library for Cellulase-ProducingColonies

The screening procedure was modified from Wood et al. (Methods inEnzymology 160, 59-74). Plates contained 20 ml 2×TY, 0.2% CMC (SigmaC-4888), 1.50% agar and 100 μg ampicillin per ml. Cells were plated inan overlay of 5 ml containing about 200 colonies per plate. The overlaywas kept at 50° C. and contains 2×TY, 0.2% CMC, 0.75% agar and 100 μgampicillin per ml. Plates were covered with 5 ml 0.5% agarose, 0.2% CMCand 100 μg ampicillin per ml kept at 50° C. after drying, the plateswere incubated for 48 hrs at 37° C. Next, 5 ml 0.1% Congo Red (Aldrichno C8, 445.3) was poured on the plates. After staining for 1-2 hrsplates were destained with 5 ml 5M NaCl for 0.5-1 hrs.

About 12.000 colonies from A. niger cDNA library (Example I) wereplated. Screening on CMC resulted in 89 colonies giving a halo afterstaining with Congo Red. Colonies were subdivided in 3 classes with alarge, intermediate and a small halo. From each class 3 colonies weregrown up, plasmids isolated and cDNAs sequenced. All contained a fulllength cDNA copy. The plasmids fell into two separate classes. From eachclass a colony was deposited at the CBS, Baarn, the Netherlands. Acolony giving a small halo was deposited on Aug. 3, 1995 and designatedCBS 589.95 (cDNA 12). A colony giving a large halo was deposited on Sep.21, 1995 and designated CBS 662.95 (cDNA 64). The DNA sequences of theinserts are presented in SEQ ID No. 1 and 3, together with the aminoacid sequences encoded by them.

The above shows that DNA fragments encoding fungal cellulases can beidentified by expression cloning in prokaryotic host cells using aplasmid vector. The following examples illustrate how cDNAs thusidentified can be used to construct strains overproducing cellulases.

Example III Overexpression of Fungal Cellulases in Aspergillus

Overexpression of cellulases may be obtained in any suitable manner,e.g., in a similar way as has been described for phytase in EP-A-0 420358 or xylanase in EP-A-0 463 706. The following elements from theseapplications are relevant for overexpression.

The A. niger amyloglucosidase (AG) promoter which ensures high levelexpression of cellulase cDNAs.

A terminator for transcription can be taken from the xylanase or phytasegenes. A sequence about 400-500 bp is sufficient for termination.

The A. nidulans AmdS marker from pGW 325 (Wernars K 1986, Thesis,Agricultural University of Wageningen, the Netherlands).

A. niger strain CBS 513.88 is a suitable receptor strain. The connectionbetween the AG promoter and the cellulase cDNAs can be made using afusion PCR (polymerase chain reaction) as described. Typically, fusionoligonucleotides are about 36 bases long and overlap both sequences atthe start codon for transcription. The fusion PCR gives rise to a DNAfragment which can be cleaved using restriction enzymes and subsequentlyligated in an E. coli vector. Similarly, a fusion experiment can beperformed fusing the stop codon for translation of the cellulase cDNAsto the terminators of xylanase or phytase. Sequences for theseterminators have been published in EP-A-0 420 358 and EP-A-0 463 706.

The fragments from the fusion PCR experiments are sequenced to check forpossible errors and subsequently the DNA fragments are ligated in aplasmid containing cellulase cDNA under control of the AG promoter and asuitable terminator. The AmdS marker can be added to this construct atany stage. All construction work is performed in E. coli.

The DNA fragment is transformed to A. niger CBS 413.88 as described inTilburn et al. (1983) Gene 26:205-221 and Kelly & Hynes (1985) EMBO J4:475-479, modified as described in EP-A-0 463 706.

Example IV Overexpression of Fungal Cellulases in K. lactis

Example IV.1

Construction of Expression Vectors

Starting vector pGBHSA20 was deposited at the CBS, Baarn, theNetherlands, on Oct. 3, 1996, and designated CBS 997.96. This vectorcontains the promoter and terminator sequence of the lactase gene (lac4)of K. lactis and a G418 selection marker. The present cDNA insertencoding HSA (human serum albumin) was replaced by cellulase encodingcDNA12 and cDNA64 from A. niger. For cloning the A. niger cDNA12, a 3′Xhol site was present. The 5′ HindIII site was created by subcloning aKpnl/EcoR1 fragment containing the full length cDNA12, in thecorresponding sites of vector pMTL22P. Digestion with HindIII, adjacentto the EcoRI site of the cDNA, and a subsequent partial digestion withXhol (cDNA12 contains an internal Xhol site) released HindIII/Xholfragment which was cloned in the unique HindIII/Xhol sites of pGBHSA20.The resulting construct, in which the HSA encoding cDNA was replaced bycDNA12, was named pCVlac12. The same cloning strategy was followed forcDNA64, resulting in expression vector pCVlac64.

Example IV.2

Transformation of K. lactis

Prior to transformation vectors pCVlac12 and pCVlac64 were linearizedwith Hpal having a unique site in the lactase promoter which is requiredfor homologous integration. K. lactis strain CBS 2359 was transformedwith 15 μg vector DNA using the LiCl method as described by Ito et al.(1983) J. Bact. 153: 163-168. Transformants were selected on YePD plates(10 g/l yeast extract, 20 g/l Bacto-peptone, 20 g/l glucose, 20 g/lBacto-agar) containing 50 μg/ml G418.

Example IV.3

Screening for Cellulase Producing K. lactis Transformants

K. lactis transformants were screened for expression of the cellulases12 and 64 in an enzyme assay using Cellazyme C tablets containingAZCL-hemicellulose colour complex (Megazyme, Australia). Cellulasesrelease a blue AZCL compound which can be quantified by measuring theabsorbance at 590 nm. To determine the cellulase activity in culturefiltrate, transformants were grown in YePD at 30° C., 200 rpm,overnight. Next day culture fluid was harvested by pelleting the cellsby centrifugation. Cellulase activity in the supernatant was variablefor independent transformants and cDNA64 containing transformants showedvery low levels of enzyme activity compared to the cDNA12 transformants.Expression of the cellulase cDNAs was enhanced when transformants weregrown on lactose instead of glucose.

Example IV.4

Mass Production of Cellulase

K. lactis transformants containing the pCVlac12 or pCVlac64 constructand with highest cellulase activity as determined in the above mentionedassays, were grown in 1 liter of YeP with 2% lactose, at 30° C., 200 rpmduring two days. Subsequently cells were pelleted by centrifugation andthe supernatant was harvested for further characterization of enzymeactivities, as described before.

Example V Characterisation of Cloned Cellulases from Aspergillus

Example V.1

Determination of Enzyme Activity

The activity of the products from pCVlac12 (L12) and pCVlac64 (L64)towards CMC, xyloglucan and β-glucan was determined by treating 250 μgof the substrate in 200 μL of a 50 mM NaOAc buffer pH 5 containing 0.01%(w/v) NaN₃ for 1 h at 40° C. The release of reducing end groups wasmeasured according to the method of Nelson-Somogyi et al. (1952) J.Biol. Chem. 195, 19-23.

CMC Xyloglucan β-glucan L12 600 29 1923 L64 458 57  729

Example V.2

Determination of pH Optimum

The pH optimum of L12 and L64 was determined using viscosity reduction.

Sample Preparation

Culture filtrate from K. lactis CBS 2359 containing (pCVlac12)T10 andK.lactis CBS 2359 containing (pVClac64)T41. Samples were concentrated byultrafiltration prior to use.

Substrates Used

Carboxymethylcellulose ([CMC] Sigma), β-glucan from barley ([βGluc]Megazyme).

Viscosimetric Method Using a “Viscorobot”

Solutions (0.4% CMC and 0.75% β-gluc) of the substrate were made inbuffers of different pH. The solutions (20 ml and up to 7 at the sametime) were placed in a temperature controlled Gilson sample changerModel 222 specially programmed for this task. The substrate solutionswere mixed with the sample (L12 or L64). Buffer was used as blank. Atregular time intervals samples were withdrawn from the mixture atconstant speed, using a Gilson Dilutor 401 controlled by the samplechanger. On withdrawal the sample was forced through a piece ofcappilair tubing, thus creating an underpressure in the system, whichwas free of any air. Pressure difference were proportional to viscosityof the sample. Pressure differences were registered by a pressuretransducer. Signals are send to an integrator which collects the data.Data is send to a computer linked to the integrator. Viscosity reductionwas plotted against time using a home made computerprogram. Relativeviscosity after a fixed time interval was plotted against the pH of thesolutions used.

FIGS. 1 and 2 show the results obtained with CMC and β-glucan. As can beseen from the figures, the CMCase pH optimum of both L12 and L64 isabout pH 3.5. As can be seen from the FIG. 2, β-glucanase of both L12and L64 is about pH 5.5

Example VI Small Scale Cloudy Apple Juice Production

After removing the peel and core, apples were homogenized in a Braunkitchen machine (MX32, Frankfurt, Germany; 5 mm blade). One g of applewas incubated (40° C.; 150 rpm) with 3 mL of a 200 mM NaOAc buffer (pH4) containing 0.01% (w/v) NaN₃, 1% (w/v) ascorbic acid, 50 mU pectinlyase and an amount of cellulase L12 preparation which was equivalent to29 mU CMCase activity. After 24 h, a cloudy juice was obtained. Thiscloud remained stable for several months.

4 1017 base pairs nucleic acid double linear cDNA NO NO Aspergillusniger N400 CBS120.49 Coding Sequence 57...773 product=“Cellulase” 1GAATTCGGCA CGAGCGAATT TCCCTTGATT GCCGCTCCTC CGCTCTAACG CCCAAC ATG 59 Met1 AAG CTC CCC GTG TCA CTT GCT ATG CTT GCG GCC ACC GCC ATG GGC CAG 107Lys Leu Pro Val Ser Leu Ala Met Leu Ala Ala Thr Ala Met Gly Gln 5 10 15ACG ATG TGC TCT CAA TAT GAC AGT GCC TCG AGC CCC CCG TAT TCA GTG 155 ThrMet Cys Ser Gln Tyr Asp Ser Ala Ser Ser Pro Pro Tyr Ser Val 20 25 30 AACCAG AAC CTC TGG GGC GAG TAC CAA GGC ACC GGC AGC CAG TGT GTA 203 Asn GlnAsn Leu Trp Gly Glu Tyr Gln Gly Thr Gly Ser Gln Cys Val 35 40 45 TAT GTCGAC AAA CTC TCC AGC AGT GGT GCA TCC TGG CAC ACC GAA TGG 251 Tyr Val AspLys Leu Ser Ser Ser Gly Ala Ser Trp His Thr Glu Trp 50 55 60 65 ACC TGGAGC GGT GGT GAG GGA ACA GTG AAA AGC TAC TCT AAC TCT GGC 299 Thr Trp SerGly Gly Glu Gly Thr Val Lys Ser Tyr Ser Asn Ser Gly 70 75 80 GTT ACA TTTAAC AAG AAG CTC GTG AGT GAT GTA TCA AGC ATC CCC ACC 347 Val Thr Phe AsnLys Lys Leu Val Ser Asp Val Ser Ser Ile Pro Thr 85 90 95 TCG GTG GAA TGGAAG CAG GAC AAC ACC AAC GTC AAC GCC GAT GTC GCG 395 Ser Val Glu Trp LysGln Asp Asn Thr Asn Val Asn Ala Asp Val Ala 100 105 110 TAT GAT CTT TTCACC GCG GCG AAT GTG GAC CAT GCC ACT TCT AGC GGC 443 Tyr Asp Leu Phe ThrAla Ala Asn Val Asp His Ala Thr Ser Ser Gly 115 120 125 GAC TAT GAA CTGATG ATT TGG CTT GCC CGC TAC GGC AAC ATC CAG CCC 491 Asp Tyr Glu Leu MetIle Trp Leu Ala Arg Tyr Gly Asn Ile Gln Pro 130 135 140 145 ATT GGC AAGCAA ATT GCC ACG GCC ACA GTG GGA GGC AAG TCC TGG GAG 539 Ile Gly Lys GlnIle Ala Thr Ala Thr Val Gly Gly Lys Ser Trp Glu 150 155 160 GTG TGG TATGGC AGC ACC ACC CAG GCC GGT GCG GAG CAG AGG ACA TAC 587 Val Trp Tyr GlySer Thr Thr Gln Ala Gly Ala Glu Gln Arg Thr Tyr 165 170 175 AGC TTC GTGTCA GAA AGC CCT ATC AAC TCA TAC AGT GGG GAC ATC AAT 635 Ser Phe Val SerGlu Ser Pro Ile Asn Ser Tyr Ser Gly Asp Ile Asn 180 185 190 GCA TTT TTCAGC TAT CTC ACT CAG AAC CAA GGC TTT CCC GCC AGC TCT 683 Ala Phe Phe SerTyr Leu Thr Gln Asn Gln Gly Phe Pro Ala Ser Ser 195 200 205 CAG TAC TTGATC AAT CTG CAG TTT GGA ACT GAG GCG TTC ACC GGG GGC 731 Gln Tyr Leu IleAsn Leu Gln Phe Gly Thr Glu Ala Phe Thr Gly Gly 210 215 220 225 CCG GCAACC TTC ACG GTT GAC AAC TGG ACC GCC AGT GTC AAC TAGGGTTCT 782 Pro AlaThr Phe Thr Val Asp Asn Trp Thr Ala Ser Val Asn 230 235 AGAAGTAGCCTTTGAGGCAG AATCTGGGTA AATTGACTCC AGCTCGGGAG AATGATAGCT 842 TGTTTCTTCGTTCTGGAACG TTGGGCGTGT GAGAGCTAAA AAGTCGTACC CACTCTGATT 902 GGAAAGACTTATTCAACATT GGTCCTTCCC TTCTGTTGGG CAAGGCATAG TTAGTGATTA 962 GACAAGTCAAGGTCATGGTG GATCCCTTGT AAAAAAAAAA AAAAAAAAAC TCGAG 1017 239 amino acidsamino acid single linear protein internal unknown 2 Met Lys Leu Pro ValSer Leu Ala Met Leu Ala Ala Thr Ala Met Gly 1 5 10 15 Gln Thr Met CysSer Gln Tyr Asp Ser Ala Ser Ser Pro Pro Tyr Ser 20 25 30 Val Asn Gln AsnLeu Trp Gly Glu Tyr Gln Gly Thr Gly Ser Gln Cys 35 40 45 Val Tyr Val AspLys Leu Ser Ser Ser Gly Ala Ser Trp His Thr Glu 50 55 60 Trp Thr Trp SerGly Gly Glu Gly Thr Val Lys Ser Tyr Ser Asn Ser 65 70 75 80 Gly Val ThrPhe Asn Lys Lys Leu Val Ser Asp Val Ser Ser Ile Pro 85 90 95 Thr Ser ValGlu Trp Lys Gln Asp Asn Thr Asn Val Asn Ala Asp Val 100 105 110 Ala TyrAsp Leu Phe Thr Ala Ala Asn Val Asp His Ala Thr Ser Ser 115 120 125 GlyAsp Tyr Glu Leu Met Ile Trp Leu Ala Arg Tyr Gly Asn Ile Gln 130 135 140Pro Ile Gly Lys Gln Ile Ala Thr Ala Thr Val Gly Gly Lys Ser Trp 145 150155 160 Glu Val Trp Tyr Gly Ser Thr Thr Gln Ala Gly Ala Glu Gln Arg Thr165 170 175 Tyr Ser Phe Val Ser Glu Ser Pro Ile Asn Ser Tyr Ser Gly AspIle 180 185 190 Asn Ala Phe Phe Ser Tyr Leu Thr Gln Asn Gln Gly Phe ProAla Ser 195 200 205 Ser Gln Tyr Leu Ile Asn Leu Gln Phe Gly Thr Glu AlaPhe Thr Gly 210 215 220 Gly Pro Ala Thr Phe Thr Val Asp Asn Trp Thr AlaSer Val Asn 225 230 235 1198 base pairs nucleic acid double linear cDNANO NO Aspergillus niger N400 CBS120.49 Coding Sequence 32...1024product=“Cellulase” 3 GAATTCGGCA CGAGATCGAG CAGTCGTAGC G ATG AAG TTT CAGAGC ACT TTG 52 Met Lys Phe Gln Ser Thr Leu 1 5 CTT CTT GCC GCC GCG GCTGGT TCC GCG TTG GCT GTG CCT CAT GGC TCC 100 Leu Leu Ala Ala Ala Ala GlySer Ala Leu Ala Val Pro His Gly Ser 10 15 20 GGA CAT AAG AAG AGG GCG TCTGTG TTT GAA TGG TTC GGA TCG AAC GAG 148 Gly His Lys Lys Arg Ala Ser ValPhe Glu Trp Phe Gly Ser Asn Glu 25 30 35 TCT GGT GCT GAA TTT GGG ACC AATATC CCA GGC GTC TGG GGA ACC GAC 196 Ser Gly Ala Glu Phe Gly Thr Asn IlePro Gly Val Trp Gly Thr Asp 40 45 50 55 TAC ATC TTC CCC GAC CCC TCG ACCATC TCT ACG TTG ATT GGC AAG GGA 244 Tyr Ile Phe Pro Asp Pro Ser Thr IleSer Thr Leu Ile Gly Lys Gly 60 65 70 ATG AAC TTC TTC CGC GTC CAG TTC ATGATG GAG AGG TTG CTT CCT GAC 292 Met Asn Phe Phe Arg Val Gln Phe Met MetGlu Arg Leu Leu Pro Asp 75 80 85 TCG ATG ACT GGT TCA TAC GAC GAG GAG TATCTG GCC AAC TTG ACG ACT 340 Ser Met Thr Gly Ser Tyr Asp Glu Glu Tyr LeuAla Asn Leu Thr Thr 90 95 100 GTG GTG AAA GCG GTC ACG GAT GGA GGC GCGCAT GCG CTC ATC GAC CCT 388 Val Val Lys Ala Val Thr Asp Gly Gly Ala HisAla Leu Ile Asp Pro 105 110 115 CAT AAC TAT GGC AGA TAC AAC GGG GAG ATCATC TCC AGT ACA TCG GAT 436 His Asn Tyr Gly Arg Tyr Asn Gly Glu Ile IleSer Ser Thr Ser Asp 120 125 130 135 TTC CAG ACT TTC TGG CAG AAT CTG GCGGGC CAG TAC AAA GAT AAC GAC 484 Phe Gln Thr Phe Trp Gln Asn Leu Ala GlyGln Tyr Lys Asp Asn Asp 140 145 150 TTG GTC ATG TTT GAT ACC AAC AAC GAATAC TAC GAC ATG GAC CAG GAT 532 Leu Val Met Phe Asp Thr Asn Asn Glu TyrTyr Asp Met Asp Gln Asp 155 160 165 CTC GTG CTG AAT CTC AAC CAA GCA GCCATT AAC GGC ATC CGC GCT GCA 580 Leu Val Leu Asn Leu Asn Gln Ala Ala IleAsn Gly Ile Arg Ala Ala 170 175 180 GGT GCA AGC CAG TAC ATT TTC GTC GAAGGC AAC TCC TGG ACC GGA GCT 628 Gly Ala Ser Gln Tyr Ile Phe Val Glu GlyAsn Ser Trp Thr Gly Ala 185 190 195 TGG ACA TGG GTC GAT GTC AAC GAT AATATG AAG AAT TTG ACC GAC CCA 676 Trp Thr Trp Val Asp Val Asn Asp Asn MetLys Asn Leu Thr Asp Pro 200 205 210 215 GAA GAC AAG ATC GTC TAT GAA ATGCAC CAG TAC CTA GAC TCC GAC GGT 724 Glu Asp Lys Ile Val Tyr Glu Met HisGln Tyr Leu Asp Ser Asp Gly 220 225 230 TCC GGC ACT TCG GAG ACC TGT GTCTCC GGG ACA ATC GGA AAG GAG CGG 772 Ser Gly Thr Ser Glu Thr Cys Val SerGly Thr Ile Gly Lys Glu Arg 235 240 245 ATC ACT GAT GCT ACA CAG TGG CTCAAG GAC AAT AAG AAG GTC GGC TTC 820 Ile Thr Asp Ala Thr Gln Trp Leu LysAsp Asn Lys Lys Val Gly Phe 250 255 260 ATC GGC GAA TAT GCC GGG GGG TCCAAT GAT GTG TGT CGG AGT GCC GTG 868 Ile Gly Glu Tyr Ala Gly Gly Ser AsnAsp Val Cys Arg Ser Ala Val 265 270 275 TCC GGG ATG CTA GAG TAC ATG GCGAAC AAC ACC GAC GTA TGG AAG GGT 916 Ser Gly Met Leu Glu Tyr Met Ala AsnAsn Thr Asp Val Trp Lys Gly 280 285 290 295 GCG TCG TGG TGG GCA GCC GGGCCA TGG TGG GGA GAC TAC ATT TTC AGC 964 Ala Ser Trp Trp Ala Ala Gly ProTrp Trp Gly Asp Tyr Ile Phe Ser 300 305 310 CTG GAG CCC CCA GAT GGA ACTGCT TAC ACG GGT ATG CTG GAT ATC CTG 1012 Leu Glu Pro Pro Asp Gly Thr AlaTyr Thr Gly Met Leu Asp Ile Leu 315 320 325 GAG ACG TAT CTC TGAGAACTGGGTGGGGTCGC AGATGCGGTG CGTCGGAGAA CTATA 1069 Glu Thr Tyr Leu 330CGGAGTTTCT TATCAGAGTG GACGGTGGTG GTACAGAGAG GCGTACTAGA ATGAATTAGT 1129GGCAGCGCAC TGACTGACGT CACAAGACAT TGCTTTTTTT GTGAAAAAAA AAAAAAAAAA 1189AAACTCGAG 1198 331 amino acids amino acid single linear protein internalunknown 4 Met Lys Phe Gln Ser Thr Leu Leu Leu Ala Ala Ala Ala Gly SerAla 1 5 10 15 Leu Ala Val Pro His Gly Ser Gly His Lys Lys Arg Ala SerVal Phe 20 25 30 Glu Trp Phe Gly Ser Asn Glu Ser Gly Ala Glu Phe Gly ThrAsn Ile 35 40 45 Pro Gly Val Trp Gly Thr Asp Tyr Ile Phe Pro Asp Pro SerThr Ile 50 55 60 Ser Thr Leu Ile Gly Lys Gly Met Asn Phe Phe Arg Val GlnPhe Met 65 70 75 80 Met Glu Arg Leu Leu Pro Asp Ser Met Thr Gly Ser TyrAsp Glu Glu 85 90 95 Tyr Leu Ala Asn Leu Thr Thr Val Val Lys Ala Val ThrAsp Gly Gly 100 105 110 Ala His Ala Leu Ile Asp Pro His Asn Tyr Gly ArgTyr Asn Gly Glu 115 120 125 Ile Ile Ser Ser Thr Ser Asp Phe Gln Thr PheTrp Gln Asn Leu Ala 130 135 140 Gly Gln Tyr Lys Asp Asn Asp Leu Val MetPhe Asp Thr Asn Asn Glu 145 150 155 160 Tyr Tyr Asp Met Asp Gln Asp LeuVal Leu Asn Leu Asn Gln Ala Ala 165 170 175 Ile Asn Gly Ile Arg Ala AlaGly Ala Ser Gln Tyr Ile Phe Val Glu 180 185 190 Gly Asn Ser Trp Thr GlyAla Trp Thr Trp Val Asp Val Asn Asp Asn 195 200 205 Met Lys Asn Leu ThrAsp Pro Glu Asp Lys Ile Val Tyr Glu Met His 210 215 220 Gln Tyr Leu AspSer Asp Gly Ser Gly Thr Ser Glu Thr Cys Val Ser 225 230 235 240 Gly ThrIle Gly Lys Glu Arg Ile Thr Asp Ala Thr Gln Trp Leu Lys 245 250 255 AspAsn Lys Lys Val Gly Phe Ile Gly Glu Tyr Ala Gly Gly Ser Asn 260 265 270Asp Val Cys Arg Ser Ala Val Ser Gly Met Leu Glu Tyr Met Ala Asn 275 280285 Asn Thr Asp Val Trp Lys Gly Ala Ser Trp Trp Ala Ala Gly Pro Trp 290295 300 Trp Gly Asp Tyr Ile Phe Ser Leu Glu Pro Pro Asp Gly Thr Ala Tyr305 310 315 320 Thr Gly Met Leu Asp Ile Leu Glu Thr Tyr Leu 325 330

What is claimed is:
 1. An isolated, purified cellulase withcarboxymethyl cellulase (CMCase), endoglucanase, β-glucanase activitiesencoded by one gene, wherein the cellulase has the amino acid sequenceSEQ ID NO:2 or SEQ ID NO:4 or a variant thereof that differs by adeletion, substitution or insertion of one or more amino acids and hasCMCase, endoglucanase and β-glucanase activities.
 2. The cellulaseaccording to claim 1 wherein the CMCase has a pH optimum of 4.5 and theβ-glucanase has a pH optimum of 5.5.
 3. The cellulase of claim 1 whereinthe gene includes the nucleotide sequence shown in either SEQ ID NO:1 orSEQ ID NO:3 or variants thereof.
 4. An enzyme composition comprising anisolated, purified cellulase with carboxymethyl cellulase (CMCase),endoglucanase and β-glucanase activities and the amino acid sequence SEQID NO:2 or SEQ ID NO:4 or variants thereof that differ by a deletion,substitution or insertion of one or more amino acids and have CMCase,endoglucanase and β-glucanase activities and a carrier.
 5. The enzymecomposition of claim 4 wherein the gene includes the nucleotide sequenceshown in either SEQ ID NO: 1 or SEQ ID NO: 3 or variants thereof.
 6. Amethod of degrading one or more of cellulose, endoglucan or β-glucan,comprising treating a source of said cellulose, endoglucan or β-glucanwith a cellulase according to claim
 1. 7. The method according to claim6 wherein said source is textile raw material.
 8. The method accordingto claim 6 wherein said source is plant raw material.
 9. A method ofdegrading one or more of cellulose, endoglucan or β-glucan, comprisingtreating said cellulose, endoglucan or β-glucan with a cellulaseaccording to claim
 1. 10. A method of degrading one or more ofcellulose, endoglucan or β-glucan, comprising treating said cellulose,endoglucan or β-glucan with an enzyme composition according to claim 4.11. A cellulase according to claim 1 wherein the cellulase is L12 orL64.